Tissue preservation system

ABSTRACT

The present invention provides a method and apparatus for tissue, such as an allograft, storage and preservation for extended periods of time at room temperature in a sterile tissue culture chamber. The invention further provides a process for maintaining the sterility of tissue using the apparatus as described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Application No.61/461,049, filed on Jan. 12, 2011, the entire disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the field of tissue, such as an allograft,storage and more specifically to the field of long-term tissue storageand preservation.

BACKGROUND OF THE INVENTION

Allograft or other tissue samples are used to treat many diseases and/ordefects. These grafts are procured from organ donors and must be storedto allow for viral and bacterial testing for safety prior to shipping tosurgical centers for implantation into patients. Based on studieslooking at viability of the cells in the grafts, recommendations havebeen given for implanting tissues as soon after harvest as possible inorder to maximize success. Safety testing takes a minimum of 7 days andmore often 10-14 days for final clearance. Storage of tissue, such asallograft tissue, for transplantation or other scientific or medicalpurposes allows time for medical testing, recipient patient preparation,or to preserve tissues for other purposes. Storage conditions forallograft or other tissue samples may influence tissue viability,integrity, and/or sterility.

SUMMARY OF THE INVENTION

Briefly described, embodiments of this disclosure provide a process andapparatus for tissue preservation. In one aspect, the invention providesa process for tissue preservation by storing the tissue at roomtemperature in a container with culture media for from about 7 to about70 days before implantation into a patient. In one embodiment, thetissue is tested for viability at least once prior to implantation in apatient. In another embodiment, viability testing is performed byassaying media that is withdrawn from the container. In anotherembodiment, cell viability is determined by adding a resazurin solutionto the media and determining the fluorescence level, wherein increasedfluorescence indicates higher cell viability.

In another embodiment, the tissue is stored in the container for from 29to about 70 days. In other embodiments, the media is changed at leastonce or about once every two weeks during storage. In anotherembodiment, at least about 70% of tissue preserved by this methodremains viable after 45 days of storage.

One aspect of the invention provides media used for storage of tissuethat is serum-free and can contain Dulbecco's Modified Eagle Medium(DMEM), high or low concentrations of glucose, antibiotic compounds(i.e., penicillin and/or streptomycin), antimycotic compounds (i.e.,Fungizone), dexamethasone, ascorbate 2-phosphate, L-proline, sodiumpyruvate, TGF-β3, and insulin, transferrin, and selenous acid, amongother chemicals or compounds.

In another aspect of the invention, the media is serum-free. In anembodiment, the media contains an effective amount of dexamethasone. Inanother embodiment, the tissue is a section of spine, scapula, humerus,radius, ulna, pelvis, femur, tibia, patella, talus, phalanges ortemporomandibular joint tissue. Other embodiments of this inventionprovide lavage of the tissue in an isotonic solution prior to storing,and implanting the tissue into a patient after storage.

Another aspect of the present invention provides a process for storageof tissue in a tissue preservation chamber containing a base, lid, mediainlet, and media outlet, wherein the media inlet is coupled to at leasta first filter for maintaining a sterile environment inside the chamber,the base is configured to contain tissue and media, the outlet extendinginto the chamber permits removal of media, a one-way valve as the mediaoutlet for removing media from the chamber, and wherein the base iscapable of receiving the lid to form a barrier to contaminants. Inembodiments of this invention, a gas exchange port is coupled to atleast a first filter, and the lid contains the media inlet, mediaoutlet, and gas exchange port. In another embodiments, the tissue isstored in the chamber for from about 29 days to about 60 days.

Another aspect of the present disclosure provides a tissue preservationchamber, including a base, lid, media inlet, and media outlet, whereinthe media inlet is coupled to at least a first filter for maintaining asterile environment inside the chamber, the base is configured tocontain cartilage tissue and media, the outlet extends into the chamberto permit removal of media, the media outlet is a one-way valve for exitof media from the chamber, and the base is capable of receiving the lidto form a barrier to contaminants. In other embodiments, a gas exchangeport is coupled to at least a first filter, the lid contains the mediainlet, media outlet and gas exchange port, and the lid is a filter thanextends across the media inlet and gas exchange port. In one embodiment,the filter is a basket adapted to be received by the lid to form arecess for sterile filter paper, the recess being in fluid and gascommunication with the media inlet and gas exchange port. In anotherembodiment, the media inlet and gas exchange port are coupled todifferent filters for maintaining a sterile environment within thechamber. In another embodiment, the media inlet and gas exchange portare coupled to the same filter to maintain a sterile environment withinthe chamber. In other embodiments, the media inlet and outlet serve asadaptors for receiving a hose. In another embodiment, the base and lidare configured to form a rim for sealing with tamper-evident tape whenin contact.

One exemplary method of tissue storage at room temperature in a chamberwith culture media before implantation includes: placing the tissue in achamber base, the chamber base configured to maintain the tissue andtissue preservation media, and forming a tissue preservation chamber bycovering the chamber base with a lid to form a barrier to contaminants,and wherein the lid contains at least one filter, a media inlet coupledto at least one filter for maintaining a sterile environment inside thechamber, and a media outlet, the media outlet including a media outletconduit that extends into the chamber to permit removal of media andreentry of media exiting the chamber. In an embodiment, the chamber alsohas a gas exchange port coupled to at least one filter. In anotherembodiment, the lid comprises the media inlet, media outlet, and gasexchange port.

An aspect of the present invention provides addition of media to thechamber through the media inlet and at least one filter. One embodimentprovides storage of the tissue in the chamber for from 29 days to about70 days. Other embodiments provide removal of the media from the chamberthrough the media outlet. A further embodiment provides simultaneousaddition of media to the chamber by forcing through the media inlet andat least one filter, along with removal of media from the chamberthrough the media outlet. Another embodiment of the present inventionprovides applying tamper evident tape to an interface between thechamber base and the lid in order to maintain sterility of the tissue.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of a tissue preservation chamberaccording to an illustrative embodiment.

FIG. 2A shows an exploded perspective view illustrating the uppersurfaces of the components of the tissue preservation chamber of FIG. 1.

FIG. 2B shows an exploded perspective view illustrating the lowersurfaces of the components of the tissue preservation chamber of FIG. 1.

FIG. 3 shows a side, cross-section view of a base of an illustrativeembodiment of a tissue preservation chamber, including a piece oftissue.

FIG. 4 shows a side, cross-section view of an illustrative embodiment ofa tissue preservation chamber, including the base of FIG. 3 and a lid.

FIG. 5 shows a side, cross-section view of an illustrative embodiment ofa tissue preservation chamber, including the flow of media into thetissue preservation chamber.

FIG. 6 shows a side view in partial cross-section of an illustrativeembodiment of a tissue preservation chamber being used to store andpreserve tissue.

FIG. 7 shows a side view in partial cross-section of an illustrativeembodiment of a tissue preservation chamber, including the flow of gasinto and out of the chamber.

FIG. 8 shows a side view in partial cross-section of an illustrativeembodiment of a tissue preservation chamber, including the flow of gasinto and out of the tissue preservation chamber and the flow of mediaout of the tissue preservation chamber.

FIG. 9 shows a side, cross-section view of an illustrative embodiment ofa tissue preservation chamber, including the flow of media into thechamber, the flow of gas into and out of the chamber, and the flow ofmedia out of the tissue preservation chamber.

FIG. 10 shows tissue viability at days 1, 28, and 56.

FIG. 11 shows tissue proteoglycan content at days 0, 28, and 56.

FIG. 12 shows tissue proteoglycan (μg GAG/mg tissue dry weight) andCollagen (μg HP/mg tissue dry weight) content at day 0 and days 63-75for each OCA storage group.

FIG. 13 shows scatter plots for each media protein biomarker (pg/ml) andthe media viability additive (fluorescence level) compared to tissueviability (LC/μm2) at the end of storage (day 63-75).

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The present disclosure provides a process and apparatus for tissuepreservation. The process includes, in one embodiment, removing viabletissue, such as allograft tissue, from a donor, testing of the tissuefor infectious diseases and/or mechanical and/or biochemical activityfor viability, placement of the viable tissue into a sterile tissueculture preservation chamber as described herein with a culture mediumcapable of maintaining the viability and sterility of the tissue, andstoring the tissue for extended periods of time prior to implantationinto a recipient. As used herein, the term “allograft” refers to atissue graft from a donor of the same species as the recipient but notgenetically identical. In an embodiment, at least about 90% of tissue orallografts preserved by the process described herein remain viable after45 days of storage. In another embodiment, the tissue is lavaged inisotonic solution prior to storing. In still another embodiment, thetissue is allograft tissue.

Allograft tissue can be removed from a donor by techniques known in theart. For instance, general aseptic surgical methods or other physicalintervention of an allograft may include but are not limited toexcision, resection, amputation, transplantation, microsurgery, generalsurgery, laser surgery, robotic surgery, or autopsy, among others.

Tissue or allograft sources may be cells, tissues, or organs from alltypes of organisms, including, but not limited to human, porcine, ovine,bovine, canine, equine, and others. In one embodiment, the source of thetissue or allograft is human. Potential allograft sources may include,but are not limited to, tissues of the eye, brain, heart, kidney, liver,intestine, bone, cartilage, skin, lung, thyroid, stomach, ligaments,tendons, or any other tissue and/or cell source that may requiretransplantation. In one embodiment of the invention, the allograft maycomprise bone and/or cartilage and/or meniscus tissue of the spine,scapula, humerus, radius, ulna, pelvis, femur, tibia, fibula, patella,talus, phalanges, or temporomandibular joint. In another embodiment, theallograft may be osteochondral tissue. Although the description hereinmay refer to allograft tissue, one of skill in the art appreciates thatother tissues find use in the method.

Once removed from the donor, the allograft is stored within the steriletissue culture chamber including, but not limited to, the chamberdescribed herein, for an extended period of time. In one embodiment, theallograft is stored at room temperature in culture media. In specificembodiments, the room temperature is between about 19° C. and 27° C.,including about 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C.,26° C., or about 26° C. In another embodiment, the allograft is storedat a temperature that is not less than about 12° C. and not more thanabout 30° C.

As used herein, the term “culture media” refers to liquid, semi-solid,or solid media used to support tissue growth and/or preservation and/ordevelopment in a non-native environment. Further, by culture media ismeant a sterile solution that is capable of stabilizing and preservingthe tissue in order to maintain its biological activity and sterility.Suitable tissue culture media are known to one of skill in the art, asdiscussed in detail subsequently. The media components can be obtainedfrom suppliers other than those identified herein and can be optimizedfor use by those of skill in the art according to their requirements.Culture media components are well known to one of skill in the art andconcentrations and/or components may be altered as desired or needed. Inone embodiment, culture media may contain Dulbecco's Modified EagleMedium (DMEM), glucose, antibiotic compounds, antimycotic compounds,dexamethasone, ascorbate 2-phosphate, L-proline, sodium pyruvate,TGF-β3, insulin, transferrin, and selenous acid, among other chemicalsor compounds. In particular, antibiotic compounds may include, but arenot limited to penicillin, streptomycin, chloramphenicol, gentamycin,and the like. Antimycotic compounds may include, but are not limited toFungizone. In an embodiment, the culture media lacks serum. Themedia-to-tissue ratio within the sterile tissue culture chamber may be10-50:1 per volume.

An unexpected benefit of the present procedure is that tissue samplescan be maintained viable and sterile for an extended period of timerelative to methods of the prior art. For instance, typically in theprior art, upon removal of an allograft from a donor, the tissue wasstored on ice or at around 4° C. Tissues prepared according to thismethod tended to remain suitably viable for around 21-28 days. However,the procedure described herein provides for a surprising and unexpectedincrease in viability of allograft tissue. Tissues prepared and storedaccording to the procedure described herein remain viable for anextended period of time relative to storage at 4° C. By an extendedperiod is meant at least between about 7-100 days, at least betweenabout 20-80 days, or at least between about 29-70, 40-70, 50-70 or 60-70days. In one embodiment an extended period is meant up to at leastaround 70 days.

It has been found that long-term storage of tissue may be facilitated byreplacement of old culture medium with fresh, sterile medium. However,prior to the present disclosure, a system that allowed for mediaexchange in an otherwise non-sterile environment was not available. Thepresent disclosure provides a system and device that allows for justthis. Advantageously, the present procedures and device provide forsterile media exchange in an otherwise non-sterile environment. Thus,media can be conveniently changed as necessary. In one embodiment, themedia is changed at least once, twice, or three times during storage.The media may be changed without removing a lid from the storagecontainer, or otherwise opening the container. The media may be changed,in specific embodiments, about once every other day, at least once aweek, at least once every two weeks, or at least about once a monthduring storage. In one embodiment, media is aspirated from the sterilechamber through a media outlet, and replaced by adding fresh mediathrough a media inlet, as described in more detail below with regard toFIG. 9. In one embodiment, a filter is placed between the lid and basechamber and media flows through the filter into the chamber. Therefore,the media remains sterile. In addition, the filter enables exchange ofCO₂ and O₂ between the chamber and the surrounding air while maintainingsterility inside the chamber.

Prior to storage according to the present disclosure, testing ofallograft tissue encompassed up to or greater than 7 days and requireddirect contact with the allograft. Such methods increased the likelihoodof allograft contamination. The present disclosure provides a convenientand easy method of testing for viability and/or contamination, by simplyextracting the culture medium from the culture chamber through the mediaoutlet, which does not compromise sterility of the tissue. The extendedstorage period allows for examination or testing of the allograft and/orculture media for a number of factors, such as viability, blood typecompatibility, HLA typing, genotyping, SNP detection, and/or infectionwith diseases. Compounds that may be detected or tested may be obtainedfrom culture media withdrawn from the sterile tested such as, but notlimited to, bacterial or virus infections, nitric oxide, prostaglandinE2, matrix metalloproteinase (MMP)-2, MMP-9, and MMP-13, vascularendothelial growth factor (VEGF), interleukin (IL)-2, IL-4, IL-6, IL-7,IL-8, IL-10, IL-15, and IL-18, granulocyte macrophage colony-stimulatingfactor (GM-C SF), Interferon gamma-induced protein (IP)-10, IFNγ,keratinocyte chemoattractant (KC), MCP-1, and TNFα. Tissue may be testedusing methods known in the art, such as by diagnostic PCR or withantibodies against biomarkers such as, but not limited to, thosedescribed above. The viability of the OCA may also be monitored duringstorage by adding a resazurin solution to the media at a finalconcentration of about 10 μg/ml and incubated at room temperature for18-24 hours. During the incubation, resazurin is converted to resorufineby viable cells in the OCA. A 200-μl sample of the media is taken andthe fluorescence level is determined using a fluorescence reader(540-570 nm excitation, 580-610 nm emission). Increased fluorescence isindicative of higher cell viability. Higher viability samples typicallyhave a fluorescence reading of ˜800-1200 units using a Synergy HT set ata sensitivity of 25 on the reader.

In view of the above, the process provides for preservation of at least70% of the allograft tissue chondrocytes after storage at roomtemperature for 45 days. In an embodiment, at least 60% or 70%, up to atleast around 99%, including 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% orgreater of the tissue is preserved when stored for 45 days, 60 days, or70 days.

In one embodiment, the process includes storing the tissue in a tissuepreservation chamber. In another embodiment, the process furtherincludes implanting the tissue in a subject in need thereof followingsaid storing.

An illustrative embodiment of a tissue preservation chamber 100 is shownin FIGS. 1, 2A, and 2B. The tissue preservation chamber 100 includes abase 102 that may be formed from any suitable material for storingtissue, e.g., an allograft. The tissue preservation chamber 100 alsoincludes a lid 106 that forms a sealed enclosure when applied to thebase. To form the sealed enclosure, the base 102 may have a firstsealing surface 140 and the lid 106 may include second sealing surface142 that abut one another when the lid 106 is applied to the base 102.In the illustrative embodiment of FIG. 1, the lid 106 is sized andconfigured to be installed to the base 102 such that the first sealingsurface 140 and second sealing surface 142 are adjacent and coplanarabout the periphery of the tissue preservation canister 100. In such anembodiment, a sterile environment may be ensured by applying a tapeabout the common surface formed at the interface of the lid 106 and base102. In an embodiment, the lid 106 may be formed with a mating feature,such as a lip on the underside of the lid 106 that forms an interferencefit with the interior surface of the base 102. In the illustrativeembodiment of FIG. 1, the lid 106 and base 102 have a rectangular shape,though it is noted that the tissue preservation chamber may be of anysuitable size and shape.

In an illustrative embodiment, the lid 106 includes features tofacilitate the controlled ingress and egress of gas and liquids to andfrom the tissue preservation chamber 100. These features include a mediaoutlet 110, media inlet 114, and gas exchange port 112. In anembodiment, the lid 106 also includes a filter basket mount 146 that maybe used to attach a filter basket 104. The filter basket mount 146 ofFIG. 2B is a molded portion of the lid 106 that includes an externalsurface 148 that mates with a complimentary internal surface 150 of thefilter basket 104. In an embodiment, the complimentary surfaces 148, 150are tapered surfaces. The filter basket 104 and base 102 may alsoinclude complementary geometrical features to locate the filter basket104 to securely hold a filter within the assembled tissue preservationchamber 100. In such an embodiment, the filter basket 104 may be mountedto the base 102 rather than the lid 106. In some embodiments, anadhesive may be used to hold the filter basket in place. As shown inFIG. 2B, the filter basket 104 includes a grated surface to support afilter without obstructing fluid flow between the tissue preservationchamber 100 and the media inlet 114 or gas exchange port 112. The filterbasket 104 of FIG. 2B is shown as having a single grated surface tosupport a single filter. Yet in some embodiments, the tissuepreservation chamber 100 may include a filter basket 104 capable ofholding multiple filters, or multiple filter baskets to facilitate theuse of separate filters for filtering flow through the media inlet 114and gas exchange port 114.

The top surface of the lid 106 includes protrusions that form the mediainlet 114, the gas exchange port 112, and media outlet 110. The mediainlet 114 may also form a similar protrusion that extends from the innersurface of the lid 106, and is located inside of the filter basket mount146 so that fluid entering the tissue preservation chamber 100 via themedia inlet 112 is routed through the filter basket 104. The gasexchange port 112 may also form a similar protrusion that extends fromthe inner surface of the lid 106, and is located inside of the filterbasket mount 146 so that fluid entering the tissue preservation chamber100 via the gas exchange port 112 is forced through the filter basket104. The protrusions that form the media inlet 114, gas exchange port112, and media outlet 110 shown in FIGS. 2A and 2B comprise taperedannular members that are suitable for coupling to, e.g., a tubingadapter. Nonetheless, the media inlet 114, gas exchange port 112, andmedia outlet 110 may have any suitable size and shape to facilitatefluid flow to and from the tissue preservation chamber 100. For example,in an embodiment, the portions of the lid 106 that protrude from the topsurface of the lid 106 to form the media inlet 114 and media outlet 110include a tapered surface to facilitate coupling to tubing adapters 118and 116, respectively. Similarly, in an illustrative embodiment, themedia outlet 110 is fluidly coupled to a media outlet conduit 108 thatextends into the tissue preservation chamber 100 to facilitate theremoval of fluid from the base of the media outlet conduit 108.

The tissue preservation chamber 100 illustrated in the Figures isoperable to store living tissue. In the illustrative embodiments, themedia outlet 110 and media inlet 114 facilitate the addition and removalof liquid, e.g., tissue culture media, to and from the tissuepreservation chamber 100 while preserving a sealed, sterile environmentwithin the tissue preservation chamber 100. Thus, once made, the tissuepreservation chamber 100 described above finds use in a method ofallograft preservation in which a tissue sample or allograft is storedin the tissue preservation chamber. Such a method is described belowwith regard to FIGS. 3-9.

In FIG. 3, tissue 138, such as an allograft, is stored in the base 102of the tissue preservation chamber 100, which is enclosed to form asealed, sterile environment. After adding the tissue 138, the lid 102and filter basket 104 may be installed to enclose the tissuepreservation chamber 100. For example, a filter 120, such as filterpaper, may be placed in the filter basket 104 that is attached to thebase 102 or filter basket mount 146 as described above. The lid 106comprises the media outlet 110, media inlet 114, and gas exchange port112. The media outlet 110 is coupled to the media outlet conduit 108,the base of which includes a media intake aperture 126 to facilitate theremoval of media from the tissue preservation chamber. To help maintainthe sterile environment within the tissue preservation chamber 100, aone-way valve 124 is affixed to the media outlet 110 to prevent theunwanted reentry of removed liquids into the tissue preservation chamber100. The gas exchange port 112 comprises an open conduit 122 that iscoupled to the filter 120, which separates the sterile environment ofthe tissue preservation chamber 100 from the external environment. Themedia inlet 114 forms a conduit through which tissue preservation mediamay be added to the tissue preservation chamber 100 after passingthrough the filter 120.

FIG. 5 shows that liquid 128, such as tissue preservation media, may beadded to the tissue preservation chamber 100 to submerge the tissue 138in the liquid 128. The liquid 128 may be added to the tissuepreservation chamber 100 through the media inlet 114 along a fluid flowpath indicated by the arrows 130. To maintain the sterile environmentwithin the tissue preservation chamber 100, the liquid 128 is forcedinto the tissue preservation chamber 100 through the filter 120.

Sealing tape 156, which may be tamper evident tape, may be applied tothe junction of the lid 106 and base 102 about the periphery of thetissue preservation chamber 100, as shown in FIG. 6. The addition ofsealing tape 156 helps to ensure the maintenance of a sterileenvironment within the tissue preservation chamber 100. The tape mayalso evidence whether the seal has been compromised. As shown in FIG. 7,tissue 138 may be stored in the tissue preservation chamber for theperiods described above. During storage, air may flow into and out ofthe chamber through the gas exchange port 112 and filter 120, asindicated by the two-way arrows 132.

The liquid 128, e.g., tissue preservation media, may be evacuated fromthe tissue preservation chamber 100 via the media outlet, as indicatedby the arrows 136 of FIG. 8. Further, constant pressure may bemaintained within the tissue preservation chamber 100 during theevacuation of fluid 128 by allowing filtered air to enter the chambervia the gas exchange port 112 as the fluid 128 is evacuated. Fluidevacuated from the tissue preservation chamber 100 is prevented fromreentering the chamber by one-way valve 124 and is removed from thesystem as indicated by arrows 134.

As shown in FIG. 9, the liquid 128 may be cycled through the tissuepreservation chamber 100 by adding liquid to the chamber through themedia inlet 114 and filter 120. Simultaneously or at another time,liquid may be removed from the tissue preservation chamber 100 via themedia outlet 110, as indicated by the arrows 134.

EXAMPLES Example 1 Analysis and Comparison of Osteochondral AllograftMetabolism Using Various Preservation Protocols

Tissue Harvest and Culture: Medial and lateral femoral condyles (FC)from both knees of 10 adult canine cadavers were aseptically harvestedwithin 4 hours of euthanasia performed for reasons unrelated to thisstudy. The volume of each FC was determined and the FCs (n=40) wereprocessed under aseptic conditions and preserved in Media 1 (M-1) (DMEM,1×ITS (insulin, transferrin, and selenous acid), non-essential aminoacids (1 mM), sodium pyruvate (10 mM), and L-ascorbic acid (50 μgimp) orMedia 2 (M-2) (DMEM, 1×ITS (insulin, transferrin, and selenous acid),non-essential amino acids (1 mM), sodium pyruvate (10 mM), L-ascorbicacid (50 μg/ml), dexamethasone (10 μM), TGF-03 (2.5 ng/ml), and sodiumborate (250 μg/ml)) at 4° C. or 37° C. for 28 or 56 days. The volume ofmedia used for preservation was determined by multiplying theapproximate volume of the tissue by 25-30. The media were changed every7 days, and samples saved for subsequent analyses. In a second study,FCs were aseptically harvested from one knee of 5 adult canine cadaverseuthanatized for reasons unrelated to this study. One FC per animal wasprocessed under aseptic conditions and preserved in Media 3 (M-3) (DMEM,1×ITS (insulin, transferrin, and selenous acid), L-glutamine (20 mM),non-essential amino acids (1 mM), sodium pyruvate (10 mM), L-ascorbicacid (50 μg/ml), penicillin, streptomycin, amphotericin B, and sodiumborate (250 μg/ml)) at 37° C. for 56 days as described above. At eachtime point, full-thickness cartilage was evaluated for tissue viability.

Media Analysis: Media were analyzed for nitric oxide (NO) by Griessassay (Promega); PGE2 by ELISA (Cayman Chemical); MMP-2, -3, -9, and -13by Luminex multiplex assay (R&D System); and IL-2, IL-4, IL-6, IL-7,IL-8, IL-10, IL-15, IL-18, GM-CSF, IP-10, IFNγ, KC, MCP-1, and TNFα byLuminex multiplex assay (Millipore).

Data Analysis: Data were compared by ANOVA and the Tukey posthoc testusing SigmaStat.

Results

Undetected Analytes: The concentration of MMP-9, IL-2, IL-4, IL-7,IL-10, IL-15, IL-18, GM-CSF, IP-10, IFNγ, and TNFα were all below thedetection level of the assay in all samples analyzed for this study,indicating little production of these proteins during culture of OCAs.

4° C. Culture vs. 37° C. Culture: After day 7, the 4° C. culture groupsreleased significantly (p<0.05) less KC, MCP-1, IL-8, MMP-2, MMP-3, andMMP-13. After day 7, production of all of these proteins decreasedsignificantly (p<0.05) in the 4° C. culture groups at all time pointstested.

The concentration of NO released to the media in the 4° C. group wassignificantly (p<0.001) lower than the M-1 and M-2 37° C. culture groupsat days 7 and 28, but not day 56. The M-3 37° C. culture group did notrelease detectable levels of NO at any time point tested, and thereforerelease significantly lower NO to the media compared to the 4° C. groupsat all time points. The concentration of PGE2 released to the media inthe 4° C. group was significantly lower than the M-1 and M-2 37° C.culture groups at all time points tested. The M-3 37° C. culture groupreleased significantly lower PGE2 to the media compared to the 4° C.culture groups at all time points.

M-1 vs. M-2 at 4° C. Culture: The M-2 4° C. culture group releasedsignificantly (p=0.008) higher NO on day 7, but not days 28 or 56,compared to the M-1 4° C. culture group. The M-1 4° C. group releasedsignificantly higher PGE2 on days 28 (p<0.001) and 56 (p=0.046), but notday 7, compared to the M-2 4° C. group. There was not a significantdifference between the M-1 and M-2 4° C. culture groups for MMP-2,MMP-3, MMP-13, KC, MCP-1, and IL-8 at any time point tested.

M-1 vs. M-2 vs. M-3 at 37° C. Culture: The M-3 37° C. culture groupreleased significantly (p<0.05) lower NO and PGE2 at all time pointstested compared to the M-1 and M-2 37° C. culture groups. The M-2 37° C.group released significantly higher NO (p=0.042) and PGE2 (p=0.046) onday 28, but not (p>0.05) days 7 and 56. At days 7 and 28, but not day56, the M-2 37° C. culture group released significantly (p<0.05) lowerMMP-2 compared to the M-1 and M-3 37° C. culture groups. At day 7, butnot days 28 and 56, the M-1 37° C. group released significantly higherMMP-3 to the media compared to the M-2 and M-3 37° C. culture groups.There was not a significant difference in the media concentration ofMMP-13 between any of the 37° C. culture groups at the time pointstested. The media concentration of KC decreased significantly over timein culture for all 37° C. groups, but there was not a significantdifference between the 37° C. culture groups at any of the time pointstested. The media concentration of IL-6 was significantly (p<0.05) lowerin the M-2 37° C. group compared to the M-1 and M-3 37° C. groups on day7, but after day 7 the media concentration of IL-6 was below the levelof detection for almost all samples. On day 7, but not days 28 and 56,the media concentration of IL-8 was significantly (p<0.05) lower in theM-2 37° C. group compared to the M-1 and M-3 37° C. groups. Further, themedia concentration of IL-8 decreased significantly (p<0.05) over timefor all 37° C. culture groups at the time points tested. At all timepoints tested, the media concentration of MCP-1 was significantly(p<0.05) higher in the M-1 37° C. group compared to the M-2 and M-3 37°C. groups. However, the media concentration of MCP-1 decreasedsignificantly (p<0.05) over time for all 37° C. culture groups at thetime points tested.

DISCUSSION

The media concentrations of the proteins analyzed in this study werevery low for tissues cultured at 4° C. after the first week of culture.This indicates that the tissue becomes quiescent under thesenon-physiologic culture conditions. Conversely, the OCAs cultured at 37°C. maintained a relatively high level of protein production indicatingthat the chondrocytes remain metabolically active during preservation.

Of the proteins analyzed, MMP-2, MMP-3, KC, MCP-1, and IL-8 wereproduced most consistently. The stable release of NO and PGE2 to themedia throughout the preservation period by tissues stored at 4° C. wasa surprising finding. Without being bound by theory, it is possible thatthe release of these two inflammatory indicators results from theprogressive cell death within the tissue and requires very littlemetabolic activity by the tissue to be produced. The NO and PGE2 dataindicate that there is a continued and stable production of theseinflammatory mediators during the preservation of the OCAs at 4 and 37°C. in M1 and M2. Importantly, the M-3 media significantly reduced themedia levels of these two inflammatory mediators, indicating that M-3may protect the tissues during culture by decreasing inflammation andpotentially improving the health of the OCA.

A potential contributing factor to failure of OCA procedures clinicallyrelates to the viability of the tissue at the time of implantation.Therefore, a biomarker assay that can differentiate between tissues withlow and high viability by testing the preservation media prior toimplantation would be of great value to tissue banks and the surgeonsusing them clinically. These data suggest that proteins evaluated inthis study are potential markers for assessment of functional viabilityof OCAs. Taken together with previous work assessing cell viability andmatrix composition of preserved OCAs, preservation of osteochondraltissues in Media 3 and 37° C. is likely to allow for preserving higherquality grafts for a longer time period than currently used protocols

Example 2 Osteochondral Allograft Preservation in a Serum-FreeChemically-Defined Media

Osteochondral allografts (OCAs) are currently preserved at 4° C. andused within 28 days of donor harvest. The window of opportunity forimplantation is limited to 14 days due to a two week disease testingprotocol, severely limiting availability to potential recipients. Thisstudy was performed to assess the effects of storage up to 56 days in aserum-free chemically defined media at 37° C. OCAs from adult caninecadavers were aseptically harvested within four hours of euthanasia.Medial and lateral femoral condyles were stored in Media 1 or 2 at 4° C.or 37° C. for up to 56 days. Chondrocyte viability, proteoglycan (GAG)and collagen (HP) content, biomechanical properties, and collagen II andaggrecan content were assessed at Days 28 and 56. Five femoral condyleswere stored overnight and assessed the next day to serve as controls.Storage in Media 1 at 37° C. maintained chondrocyte viability atsignificantly higher levels than in any other media-temperaturecombination examined and at levels not significantly different fromcontrols.

OCAs stored in either media at 4° C. showed a significant decrease inchondrocyte viability throughout storage. GAG and HP content weremaintained through 56 days of storage in OCAs in Media 1 at 37° C. Therewere no significant differences in elastic or dynamic moduli amonggroups at Day 56. Qualitative immunohistochemistry demonstrated thepresence of collagen II and aggrecan throughout all layers of cartilageduring storage. OCA viability, matrix content and composition, andbiomechanical properties were maintained at “fresh” levels through 56days of storage in media 1 at 37° C. OCAs stored at 4° C. were unable tomaintain viability or matrix integrity through 28 days of storage.

Storage Protocol:

OCAs within 4 hours of death from medial and lateral femoral condyles ofadult canine cadavers euthanized for reasons unrelated to this study.Allografts were stored overnight in media at 37° C., 95% humidity, and5% CO2. Day 0 Control OCAs (n=5) were aseptically harvested from onefemoral condyle of 5 adult canine cadavers euthanized for reasonsunrelated to this study. These OCAs were stored overnight in serum-freemedia and evaluated the following day.

The volume of each OCA (n=40) was determined and storage media volumesused were 25-30 times OCA volume. The OCAs were stored in Media 1 (DMEM,1×ITS (insulin, transferrin, and selenous acid), non-essential aminoacids (1 mM), sodium pyruvate (10 mM), and L-ascorbic acid (50 μg/ml))or Media 2 (DMEM, 1×ITS (insulin, transferrin, and selenous acid),non-essential amino acids (1 mM), sodium pyruvate (10 mM), L-ascorbicacid (50 μgimp, dexamethasone (10 μM), TGF-β3 (2.5 ng/ml), and sodiumborate (250 m/ml)).

Once the OCAs were aseptically processed they were preserved in eitherMedia 1 or 2 at 4° C. or 37° C. for 28 or 56 days. Media 1 was designedto provide basic nutrition to the tissue and Media 2 was designed to beanti-inflammatory and chondrogenic. Each stored specimen had its owncontralateral control on the opposite leg. The media were changed every7 days and media samples were saved for subsequent analyses. At eachtime point full-thickness cartilage was evaluated for chondrocyteviability, biochemical composition, and biomechanical properties.

Tissue Viability Analysis:

Full-thickness cartilage from each storage group was used to determinechondrocyte viability. Quantitative analysis of chondrocyte viabilitywas determined by manually counting live and dead cells from images forthe entire sample taken at 10× magnification using an Olympus F view IIcamera and Micro Suite Basic Edition software. CellTracker™ Green CMFDA(5-chloromethylfluorescein diacetate, Invitrogen, Carlsbad, Calif.) wasused to visualize live cells and ethidium homodimer-1 (EthD-1,Invitrogen, Carlsbad, Calif.) to visualize dead cells. Percent livecells were determined by taking the total number of live cells dividedby the total amount of cells in the sample.

GAG Content:

After PBS wash, cartilage plugs were blotted with a paper wipe andweighed on balance to obtain the wet weight. Dry weight was determinedafter lyophilization for 24 hours. Cartilage tissue was the thendigested in 504, papain solution for 3 hours at 60° C. Sulfated-GAG(s-GAG) concentration within the cartilage matrix was determined usingaliquots of digest solution using the 1,9 dimethylmethylene blue (DMMB)dye-binding assay. S-GAG content was determined from the ratio of s-GAGto total tissue dry weight and reported at μg GAG/mg dry weight.

HP Content:

The hydroxyproline (HP) content was determined using a colorimetricassay modified to a 96-well format. HP content was used as a measure oftotal collagen content. A 50-μL aliquot of the digest solution was mixedwith 50 μL 4N NaOH and the mixture was autoclaved for 20 minutes at 121°C. to hydrolyze the sample. The sample was then mixed with thechloramine T reagent and incubated at 25° C. for 25 minutes, followed bymixing with Ehrlich aldehyde reagent. The chlorophore was developed at65° C. for 20 minutes. The absorbance was then read at 550 nm using aSynergy HT (Bio-TEK, Highland Park, Vt.) and the samples were comparedwith an HP standard to determine the HP concentration of the sample.Results were standardized to tissue dry weight and reported as μg HP/mgdry weight.

Biomechanical Analysis:

At each end point, 4-mm plugs were removed from the articular cartilageand immediately put in a −80° C. freezer until biomechanical testingcould be done. The dynamic modulus of cartilage specimens weredetermined by unconfined compression with loading to 10% strain at arate of 0.05% per second, after an initial 0.02-N tare load (elasticmodulus, or Eγ). Dynamic modulus (G*) was measured by superimposing 2%peak-to-peak sinusoidal strain at 0.1 Hz. Values were reported asmegapascals (MPa).

Immunohistochemical Analysis:

For immunohistochemical evaluation, 2-mm sagittal sections of OCAs werecut and fixed in 10% formalin. After fixation was complete, samples weredecalcified in 10% disodium ethylenediaminetetraacetic (EDTA) acid.After decalcification and subsequent routine histologic processing, eachspecimen was embedded in paraffin, and sectioned 5 μm through thesagittal plane. For immunohistochemical analysis, unstained sectionswere deparaffinized in xylene and rehydrated in graded ethanolsolutions. The samples were permeabilized with a 0.1% trypsin solutionat 36° C. for 60 minutes and then blocked with a 10% bovine serumalbumin at 40° C. Slides were incubated overnight at 4° C. inpredetermined dilutions of the primary antibodies: collagen type II(rabbit polyclonal antibody, Abcam, Cambridge, UK) and proteoglycan(mouse anti-human antibody, Millipore Corp., Billerica, Mass.).

The next day, slides were rinsed in Tris-buffered saline before beingincubated with the secondary antibody. Collagen type II was labeled withgoat anti-rabbit fluorescein isothiocyanate (FITC, Millipore Corp.,Billerica, Mass.) and proteoglycan was labeled with goat anti-mouserhodamine (Millipore Corp., Billerica, Mass.). Samples were coverslippedand reviewed using fluorescent light microscopy. Negative controls wereused as comparison in which the primary (but not secondary antibody) wasomitted from the slides to see if any stain was due to fluorescenceaside from the target region. Immunohistochemical images weresubjectively assessed.

Statistical Analyses:

Statistical analyses were done using the SigmaStat® computer softwareprogram (San Rafael, Calif.). Data were pooled for each endpoint, Day 28and Day 56, and comparisons were made among the four storage media andDay 0 controls. A one way ANOVA using Tukey post-hoc comparisons wasused for statistical analysis with significance set at p<0.05.

Results:

Chondrocyte viability of femoral condyle OCAs stored in Media 1 at 37°C. was significantly higher than Media 1 and 2 at 4° C. (p=0.016,p=0.01) at Day 28. At Day 56, Media 1 at 37° C. had significantly highercell viability than Media 1 at 4° C. (p=0.008) and Media 2 at 4° C. and37° C. (p=0.015, p=0.023). When comparing stored OCAs to Day 0 controls,controls had significantly higher viability than OCAs in Media 1 and 2at 4° C. (p=0.007, p=0.01) at Day 28. OCAs stored in Media 1 at 37° C.were the only group able to maintain viability at levels notsignificantly different that controls through Day 56. Chondrocyteviability in control OCAs was significantly higher than OCAs in Media 1at 4° C. (p=0.032) and Media 2 at 4° C. (p<0.001) and 37° C. (p=0.002)at Day 56.

Analysis of tissue GAG content of femoral condyle OCAs showed nosignificant differences among storage groups at Day 28. At Day 56, OCAsstored in Media 2 at 37° C. had significantly less tissue GAG contentthan Media 1 at 4° C. (p=0.027) and 37° C. (p=0.033). At Day 28, therewere no significant differences in tissue GAG compared to controls.However, at Day 56, controls had significantly more tissue GAG contentthan OCAs stored in Media 2 at 37° C. (p=0.003).

There were no significant differences among femoral condyle OCA storagegroups with respect to HP content at Days 28 or 56. Also, there were nosignificant differences at any time point compared to controls.Biomechanical analyses of femoral condyle OCAs showed elastic modulus ofcontrols to be significantly higher than OCAs in Media 1 at 37° C.(p=0.017) and Media 2 at 4° C. (p=0.016) at Day 28. Dynamic modulus wassignificantly higher in controls than OCAs in Media 1 at 4° C. (p=0.032)and 37° C. (p=0.022) as well as Media 2 at 4° C. (p=0.041) at Day 28.There were no significant differences noted for Day 56 analyses.

Example 3 Assessment of Potential Biomarkers for Evaluating Viability ofOsteochondral Allograft Tissue During Preservation

Osteochondral allografts (OCA) allow transplantation of viable,functional tissue for treatment of cartilage defects without the needfor immunosuppression. OCAs are reported to be successful in >75% ofcases when used for treatment of focal femoral condyle lesions. Thepresent study was designed to evaluate the ability of biomarkers todifferentiate OCAs with low viability during culture using varioustissue preservation protocols.

Tissue Harvest and Culture:

Medial and lateral femoral condyles (FC) from both knees of 10 adultcanine cadavers were aseptically harvested within 4 hours of euthanasiaperformed for reasons unrelated to this study. The volume of each FC wasdetermined and the FCs (OCAs, n=40) were processed under asepticconditions and preserved in Media 1 (M-1) or Media 2 (M-2) at 4° C. or37° C. for 28 or 56 days. The volume of media used for preservation wasdetermined by multiplying the approximate volume of the tissue by 25-30.The media were changed every 7 days, and collected for analysis ofbiomarker production. In a second study, the FCs were asepticallyharvested from one knee of 4 adult canine cadavers euthanatized forreason unrelated to this study.

One FC per animal was processed under aseptic conditions and preservedin Media 3 (M-3) (DMEM, 1×ITS (insulin, transferrin, and selenous acid),L-glutamine (20 mM), non-essential amino acids (1 mM), sodium pyruvate(10 mM), L-ascorbic acid (50 μg/ml), penicillin, streptomycin,amphotericin B, and sodium borate (250 μg/ml)) at 37° C. for 56 days asdescribed above. At each time point, full-thickness cartilage wasevaluated for tissue viability.

Tissue Viability Analysis:

Cartilage tissue was analyzed for cell viability using a fluorescentlive/dead assay (Invitrogen) and fluorescent microscopy. Images weretaken at 10× magnification using an Olympus F-View II camera andMicroSuite Basic Edition software. For the M-1 and M-2 samples, twotissue sections were used for evaluation of tissue viability. For theM-3 samples at least three tissue sections were taken for evaluation oftissue viability. Green-staining live cells were manually counted andthe area of the tissue analyzed was determined using MicroSuite BasicEdition. Because % cell viability does not take into account total lossof cells, the area of the tissue section analyzed was measured, and theratio of live cells (LC)/area (mm2) was determined.

Media Analysis:

Media were analyzed for MMP-2, -3, and -13 (R&D System) and IL-8, KC,and MCP-1 (Millipore) using two Luminex multiplex assay.

Data Analysis:

Data were compared by Pearson product-moment correlation usingSigmaStat.

Tissue Viability (Table 1):

The mean tissue viability represented the viability of each group well,but each group had one outlier. Further, the viabilities of M-2-37 andM-3-37 were significantly higher than all other groups. Because theresponse of the OCAs to each preservation protocol was unique, the mediaprotein data were analyzed to determine if the outliers could beidentified in each group.

All 4° C.

Culture Groups: After day 14, the only analytes tested that wereconsistently detected were MMP-3 and KC, and the concentrations of theseproteins were significantly lower than the day 7 values at all timepoints. Further, a difference in the media concentration could not bedetermined between the OCAs with the lowest tissue viability (0.23LC/mm2 M-1, 0.093 LC/MM2 M-2) and the highest tissue viability (1.14LC/MM2 M-1, 0.747 LC/MM2 M-2).

M-1 37° C. Culture Groups:

The variability in the tissue viability of the M-1 group was relativelylow, and the viability of the tissues was relatively high. Therefore,there was not a distinct difference in the biomarker values of thesamples with low viability (˜0.7 LC/mm2) and high viability (>1.0LC/mm2). Interestingly, there was a negative weak to moderatecorrelation between cell viability and all the biomarkers analyzed inthis study.

M-2 37° C. Culture Groups:

The tissue viability of this group was significantly lower than theother 37° C. groups. After day 7 the concentration of KC and IL-8 hadmoderate-strong positive correlations (0.569-0.995) with tissueviability depending on the day analyzed. MCP-1 had weak-moderate(0.339-0.613) positive correlations with tissue viability throughout theculture period. MMP-2, MMP-3, and MMP-13 all had a moderate-strong(0.651-0.927) positive correlations on days 7 and 28, and aweak-moderate (0.337-0.6) positive correlations on day 56. The twosamples with the lowest tissue viability (<0.1 LC/mm2) had little to nodetectable KC, IL-8, MCP-1, MMP-2, MMP-3, and MMP-13 after day 7.

M-3 37° C. Culture Groups:

The M-3 group had the largest disparity between the samples with thehighest (>1.0 LC/mm2) viability (n=3) and lowest (0.00 LC/mm2) viability(n=1). After day 14, little to no MCP-1, IL-8, and KC detectable in themedia of the low viability sample. Early in culture, KC and MCP-1 hadstrong (0.815-0.972) positive correlations to tissue viability, and atlater time points in culture each had moderate (0.5-0.78) positivecorrelations to tissue viability. At all time points, IL-8 had moderate(0.566-0.751) positive correlations to cell viability. MMP-2 had strong(0.811-0.907) positive correlations through day 21; MMP-3 had strong(0.889-0.968) positive correlations after day 7; and MMP-13 had weak(0.3-0.461) positive correlations to tissue viability at all timepoints. The sample with the lowest tissue viability (0.00 LC/mm2) hadlittle to no detectable KC, IL-8, and MCP-1 after day 7, but MMP-2,MMP-3, and MMP-13 could be detected at all time points.

These data indicate that proteins in the preservation media have thepotential to act as biomarkers for distinguishing OCAs that have verylow cell viability and therefore are not considered suitable forclinical use. If implemented, tissue banks could readily and repeatedlyassess the usefulness of the tissue during the preservation periodwithout the need for sectioning the grafts. This would essentially allowtissue banks to cull samples as soon as they are no longer acceptablefor clinical use, saving time and expense. It would also allow surgeonsto have more confidence in the quality of the grafts that they areimplanting into patients. KC, MCP-1, and MMP-3 are the strongestcandidate biomarkers to identify OCAs with low tissue viability duringculture.

TABLE 1 Tissue viability for each tissue preseravtion protocol StorageTemp Days in Tissue Viability (LC/mm2) Media (C.) Storage Mean Range M-14 28 0.57 0.245-1.088 M-1 4 56 0.41 0.112-1.143 M-1 37 28 1.050.819-1.24  M-1 37 56 1.03 0.736-1.32  M-2 4 28 0.5 0.033-0.892 M-2 4 560.37 0.093-0.747 M-2 37 28 0.6 0.110-0.882 M-2 37 56 0.36 0.009-0.628M-3 37 56 1.32 0.00-1.44

Example 4 Optimization of Osteochondral Allograft Preservation to Extendthe Usable Life Span of Harvested Tissue

The present study was designed to evaluate the effectiveness ofculturing OCAs at 37° C. using different media compositions forextending the pre-implantation life span of harvested tissue based ontissue viability and matrix composition.

Methods Tissue Harvest and Culture:

Medial and lateral femoral condyles (FC) from both knees of 10 adultcanine cadavers were aseptically harvested within 4 hours of euthanasiaperformed for reasons unrelated to this study. The volume of each FC wasdetermined and the FCs (OCAs, n=40) were processed under asepticconditions and preserved in Media 1 (M-1) or Media 2 (M-2) at 4° C. or37° C. for 28 or 56 days. The volume of media used for preservation wasdetermined by multiplying the approximate volume of the tissue by 25-30.The media were changed every 7 days, and saved for subsequent analyses.In a second study, FCs were aseptically harvested from one knee of 5adult canine cadavers euthanatized for reason unrelated to this study.One FC per animal was used as a freshly harvested day 0 control (n=5),and the other was processed under aseptic conditions and preserved inMedia 3 (M-3) (DMEM, 1×ITS (insulin, transferrin, and selenous acid),L-glutamine (20 mM), non-essential amino acids (1 mM), sodium pyruvate(10 mM), L-ascorbic acid (50 μg/ml), penicillin, streptomycin,amphotericin B, and sodium borate (250 μg/ml)) at 37° C. for 56 days asdescribed above. At each time point, full-thickness cartilage wasevaluated for tissue viability, proteoglycan (GAG) content, and collagen(HP) content.

Viability Analysis:

Cartilage tissue was analyzed for cell viability using a fluorescentlive/dead assay (Invitrogen) and fluorescent microscopy. For the M-1,M-2, and Day 0 control tissues full thickness cartilage was excised fromthe bone; two 4 mm cartilage plugs were created from the tissue using adermal punch; a ˜0.5 mm thick slice was taken from the middle of theplug, and the slice was stained for 30 minutes at 37° C. For the M-3tissues a diamond saw was used to make a 0.5 mm section from the centerof the FC and this section was then stained for 30 minutes at 37° C.Images were taken at 10× magnification using an Olympus F view II cameraand MicroSuite Basic Edition software. For the M-1, M-2, and Day 0samples one image from each slice (n=2 images) was used for evaluationof tissue viability. For the M-3 samples, at least 3 images fromdifferent areas of the slice were used for evaluation of tissueviability. Greenstaining live cells were manually counted and the areaof the tissue analyzed was determined using MicroSuite Basic Edition.Because % cell viability does not take into account total loss of cells,the area of the tissue section analyzed was measured, and the ratio oflive cells (LC)/area (mm2) was determined.

Biochemical Analyses:

Tissue GAG content was determined using the dimethylmethylene blueassay. Tissue HP content was determined using the hydroxyproline assay.Tissue GAG and HP content was standardized to tissue dry weight.

Data Analysis:

Data were compared by ANOVA and the Tukey post-hoc test using SigmaStat.

Results Tissue Culture:

One sample in the M-2-37-56 group and M-3-37-56 group was lost toprocessing problems. Therefore, these groups only had 4 samples foranalysis.

Tissue Viability (FIG. 10):

The mean tissue viability of the day 0 controls was 1.13 LC/mm2(1.03-1.25 LC/mm2). The tissue viability of the M-1-37 group was notsignificantly different than day 0 group at day 28 or 56. There was nota significant difference between the M-3-37-56 group and the day 0control for tissue viability. The M-1-4, M-2-4, and M-2-37 groups allhad significantly lower tissue viability compared to the day 0 control(p<0.005-0.008), the M-1-37 group (p<0.016-0.025), and M-3-37 group(p<0.004-0.006) at all time points. There was not a significantdifference between the M-1-37 and M-3-37 groups.

Tissue Matrix Composition:

On day 56 the M-2-37 group had significantly (p<0.003-0.027) lowertissue GAG content compared to the day 0, both M-1 groups, and theM-3-37 group (FIG. 11). Further, the M-2-4 group had significantly(p<0.01) lower tissue GAG content compared to the M-3-37-56 group. Therewere no other significant differences for tissue GAG content. There wasnot a significant difference in the collagen content of the tissuesbetween any groups at any time point based on HP analysis.

Example 5 Analysis of Osteochondral Allograft Metabolism Using VariousPreservation Protocols at 25° C.

Osteochondral allografts (OCA) allow transplantation of viable,functional tissue for treatment of cartilage defects without the needfor immunosuppression. Currently, tissue banks store OCAs at 4° C. andrecommend implantation within 28 days of harvest. The present study wasdesigned to evaluate the effects of various tissue preservationprotocols on the metabolism of OCAs based on the release of degradativeenzymes, cytokines, and chemokines to the media at 25° C. previouslyshown to be released during 37° C. storage.

Methods Tissue Harvest and Culture:

During the course of two studies, medial and lateral femoral condyles(FC) from both knees of 14 adult canine cadavers were asepticallyharvested within 4 hours of euthanasia performed for reasons unrelatedto this study. The FCs were separated into one of 5 test groups based ondifferent media composition (M-1 (DMEM, 1×ITS (insulin, transferrin, andselenous acid), L-glutamine (20 mM), non-essential amino acids (1 mM),sodium pyruvate (10 mM), L-ascorbic acid (50 μg/ml), penicillin,streptomycin, and amphotericin B), M-2 (DMEM, 1×ITS (insulin,transferrin, and selenous acid), L-glutamine (20 mM), non-essentialamino acids (1 mM), sodium pyruvate (10 mM), L-ascorbic acid (50 μg/ml),penicillin, streptomycin, amphotericin B, and sodium borate (250μg/ml)), M-3 (DMEM, 1×ITS (insulin, transferrin, and selenous acid),L-glutamine (20 mM), non-essential amino acids (1 mM), sodium pyruvate(10 mM), L-ascorbic acid (50 μg/ml), penicillin, streptomycin,amphotericin B, and dexamethasone (0.01 μM))) and container condition(C-1, C-2, C-3) such that each FC from a single animal was placed in adistinct group. The following media and container condition groupingswere assessed for this study M-1/C-1, M-1/C-2, M-1/C-3, M-2/C-1, andM-3/C-3, resulting in the 5 different OCA storage groups. Tissues werestored at 25° C. without CO2 supplementation in 60 mls of media for atleast 63 days and up to 75 days. The media were changed every 7 days andsaved for biomarker analyses.

Media Analysis:

Media were analyzed for VEGF, matrix metalloproteinase (MMP)-2, -3, -9,and -13 by Luminex multiplex assay (R&D System); and IL-6, IL-8, KC, andMCP-1 by Luminex multiplex assay (Millipore).

Data Analysis:

Data from days 7, 28, and 56 of storage were compared by ANOVA and theTukey post-hoc test using SigmaStat.

Results

MMP-2: The release of MMP-2 to the media increased in all groups afterday 7, and remained stable from day 14 to the end of storage in theM-1/C-2 and M-1/C-3 groups of study 1 and the M-1/C-1, M-1/C-3, andM-3/C-3 of study 2. The M-1/C-1 and M-2/C-1 groups in study 1 haddecreasing levels of MMP-2 released to the media over time in cultureafter day 21, and on days 28 and 56 the M-1/C-3 had significantly highermedia MMP-2 levels compared to the M-1/C-1, M-1/C-2, and M-2/C-1 groups.In study 2 the M-3/C-3 group had significantly lower MMP-2 compared tothe M-1/C-3 group on days 28 and 56, and the M-1/C-1 group on day 28.MMP-3: After day 7 all groups in study 1 and the M-1/C-1 group in study2 released decreasing levels of MMP-3 to the media. However, the levelof MMP-3 in the M-1/C-3 group of study 1 remained stable after day 14and throughout culture for the M-1/C-3 and M-3/C-2 group in study 2. Onday 28 of study 1, the M-2/C-1 group had significantly lower media MMP-3levels compared to the M-1/C-1 and M-1/C-3 group, and on day 56 of study1 the M-1/C-3 group had significantly higher media MMP-3 levels comparedto all other groups. In study 2 the level of MMP-3 was not significantlydifferent between any groups at the time points analyzed.MMP-9: MMP-9 was not detected at any time point tested in both study 1and study 2.MMP-13: In study 1 the level of MMP-13 in the media increased quicklyafter day 7 in the M-1/C-1 and M-1/C-2 groups, and more slowly in theM-1/C-3 and M-2/C-1 groups. On day 28 the M-1/C-3 group hadsignificantly lower media MMP-13 compared to the M-1/C-1 and M-1/C-2groups. In study 2 the M-3/C-3 group had significantly lower mediaMMP-13 levels compared to the M-1/C-1 and M-1/C-3 groups on day 7 and28, and the M-1/C-1 group on day 56.KC: In study 1 the level of KC in the media was stable in the M-1/C-3,but decreased significantly in all other groups over time in storage. Instudy 2 the level of KC decreased significantly after day 7 in allgroups over time. In study 1, the media level of KC in the M-2/C-1 groupwas significantly lower than the M-1/C-2 and M-1/C-3 groups on day 7,all groups on days 28 and 56. In study 2, the media level of KC wassignificantly lower than in the M-3/C-3 group compared to the M-1/C-3group on days 28 and 56.IL-6: In study 1 and study 2 the media level of IL-6 spiked at day 7 anddecreased significantly and rapidly in all samples on subsequent days.In study 1 the M-2/C-1 group had significantly lower media IL-6 on day 7compared to all other groups, and the M-1/C-3 group had significantlyhigher IL-6 on day 56 than all other groups. In study 2 the M-3/C-3group had significantly lower media IL-6 compared to the M-1/C-1 andM-1/C-3 group on day 28 and 56.IL-8: In study 1 the media level of IL-8 was relatively stable over timein all groups but the M-2/C-1 group, which had decreasing medial IL-8levels over time in storage. In study 2 the level of IL-8 decreased overtime in culture in all groups. In study 1 the M-2/C-1 group hadsignificantly lower media IL-8 levels compared to all groups at all timepoints analyzed. Further, the M-1/C-3 group had significantly highermedia IL-8 levels compared to all other groups on day 56. In study 2 theM-3/C-3 group had significantly lower media IL-8 levels compared to theM-1/C-3 group at all time points and the M-1/C-1 group on days 28 and56.MCP-1: In study 1 the media level of MCP-1 decreased after day 14 andstabilized in all groups but the M-2/C-1 group, which had low MCP-1media levels from day 7 through day 56 of culture. In study 2 the medialevel of MCP-1 decreased after day 28 in culture in all groups. In study1 the media concentration of MCP-1 in the M-2/C-3 group wassignificantly lower than all other groups on days 28 and 56. Further,the M-1/C-3 group had significantly higher media MCP-1 compared to theM-1/C-1 group on days 28 and 56. In study 2 the media concentration ofMCP-1 in the M-3/C-3 group was significantly lower than the M-1/C-3group on day 56 of storage.VEGF: In study 1 the media level of VEGF was stable over time in theM-1/C-3 group, but decreased over time in all other groups. In study 2the level of VEGF was stable in all groups over time in culture. Instudy 1 the M-2/C-1 group had significantly lower media VEGFconcentrations compared to all groups on day 7 and the M-1/C-2 andM-1/C-3 groups on day 56. In study 2 the M-3/C-3 group had significantlylower media VEGF concentrations compared to the M-1/C-3 group at alltime points and the M-1/C-1 group on days 7 and 28.

Discussion

These data indicate that OCA tissues are metabolically active during 25°C. storage, and that the same proteins detected in previous studies at37° C. storage are also detected at 25° C. storage. Further, the patternof production of these biomarkers at 25° C. is similar to that observedat 37° C.

Example 6 Optimization of Long-Term Osteochondral Allograft Storage at25° C.

Osteoarthritis (OA) affects ˜90% of people older than 65, and associatedcosts top $100 billion annually in the U. S. One treatment available forfocal lesions is osteochondral allografts (OCA) transplantation. OCAsare reported to be successful in >75% of cases when used for treatmentof focal femoral condyle lesions. Currently, tissue banks store OCAs at4° C., and implantation is recommended within 28 days after harvest dueto significant loss in tissue viability by this time point. Becausemandatory disease screening protocols typically take 14 days tocomplete, the window for surgical implantation is narrow (˜14 days),which severely limits clinical use. Therefore, this study was designedto evaluate the effectiveness of culturing OCAs at 25° C. using novelmedia compositions and container conditions for extending thepre-implantation life span of harvested tissue based on tissue viabilityand matrix composition.

Methods: Tissue Harvest and Culture:

During the course of two studies, medial and lateral femoral condyles(FC) from both knees of 14 adult canine cadavers were asepticallyharvested within 4 hours of euthanasia performed for reasons unrelatedto this study. The FCs were either used as day 0 controls (n=7) orseparated into one of 5 test groups based on different media composition(M-1 (DMEM, 1×ITS (insulin, transferrin, and selenous acid), L-glutamine(20 mM), non-essential amino acids (1 mM), sodium pyruvate (10 mM),L-ascorbic acid (50 μg/ml), penicillin, streptomycin, and amphotericinB), M-2 (DMEM, 1×ITS (insulin, transferrin, and selenous acid),L-glutamine (20 mM), non-essential amino acids (1 mM), sodium pyruvate(10 mM), L-ascorbic acid (50 μg/ml), penicillin, streptomycin,amphotericin B, and sodium borate (250 μg/ml)), M-3 (DMEM, 1×ITS(insulin, transferrin, and selenous acid), L-glutamine (20 mM),non-essential amino acids (1 mM), sodium pyruvate (10 mM), L-ascorbicacid (50 μg/ml), penicillin, streptomycin, amphotericin B, anddexamethasone (0.01 μM))) and container condition (C-1, C-2, C-3) suchthat each FC from a single animal was placed in distinct group. Thefollowing media and container condition groupings were assessed for thisstudy M-1/C-1, M-1/C-2, M-1/C-3, M-2/C-1, and M-3/C-3, resulting in the5 different OCA storage groups. Tissues were stored at 25° C. withoutCO2 supplementation in 60 mls of media for at least 63 days and up to 75days. The media were changed every 7 days and saved for biomarkeranalyses. At the end of storage, osteochondral plugs were evaluated fortissue viability and matrix composition.

Viability Analysis:

Cartilage tissue was analyzed for cell viability using a fluorescentlive/dead assay (Invitrogen) and fluorescent microscopy. Osteochondraltissues were incubated in stain for 25 minutes at 25° C. Images weretaken at either 4× (study 1) or 10× (study 2) magnification.Green-staining live cells were manually counted, and the area of thetissue analyzed was determined. The viability of the tissue is expressedas the ratio of live cells (LC)/area (μm²). Because the focal depth of4× images was significantly different than the focal depth of 10×images, the viability could not be compared between the 4× and 10×images, and analysis was only performed between samples that were takenat the same magnification.

Matrix Composition:

Cartilage tissue was lyophilized and weighed, digested with papain, andanalyzed for proteoglycan content using the DMMB assay and collagencontent using a hydroxyproline assay. GAG and collagen content wasnormalized to tissue dry weight.

Data Analysis:

Data were compared by ANOVA and the Tukey post-hoc test using SigmaStat.

RESULTS: Day 0 and Day 63-75 Tissue Viability (Table 2):

The mean tissue viability and range are listed for each group at day 63for each magnification. For the samples analyzed at 4× magnification,day 0 and the M-3/C-3 group had significantly higher tissue viability(LC/mm2) compared to the M-1/C-1 and M-2/C-1 groups at day 63, and theM-1/C-3 group had significantly higher tissue viability compared to theM-2/C-1 group at day 63. The M-3/C-3 group had the highest meanviability and the lowest variability of all the storage groups tested.The sample size was smaller for the 10× magnification groups, and therewas not a significant difference between the groups as seen in the firstset of samples analyzed at 4× magnification. However, in agreement withthe 4× data, the M-3/C-3 group had the highest viability with the lowestvariability and was closest to day 0 viability values.

Day 0 and Day 63-75 Cell Distribution:

The day 0 and M-3/C-3 groups had good cell numbers distributed throughthe thickness of the tissue. The M-1/C-1 and M-1/C-2 groups typicallyhad very low cell numbers in the superficial-middle zones of the tissueand higher cell numbers in the deep-middle zones of the tissue. TheM-2/C-1 group had very few detectable viable cells in any region of thetissue.

Matrix Composition (FIG. 12):

The proteoglycan content of the M-3/C-3 group was significantly lowerthan the day 0 and all other storage groups. The GAG content of thetissues was not a significantly different between any other groups inthis study. The HP content of the first set of tissues stored could notanalyzed for HP content, so there is no HP data for the M-1/C-2 andM-2/C-1 groups tissues. Of the samples tested, there was not asignificant difference between the groups tested.

Discussion:

These data indicate that femoral condyle OCA tissue stored at 25° C.without CO2 supplementation can maintain day 0 tissue viability up to 75days in storage. This is a significant improvement over currentprotocols at 4° C., which shows significant loss of tissue viability byday 28 of storage.

TABLE 2 Tissue viability for each tissue protocol 4x Tissue Viability10x Tissue Viability CONTAINER (LC/μm2) (LC/μm2) Media CONDITION MeanRange Mean Range M-1 C-1 0.916 0.038-3.24  0.623  0.0-1.38 M-1 C-2 2.1151.29-2.62 M-1 C-3 2.217  0.1-3.49 0.804 0.213-1.36  M-2 C-1 0.0423 0.0-0.117 M-3 C-3 3.195 2.99-3.31 1.137 0.97-1.29 Day 0 2.901  0.5-5.351.13 1.02-1.25

Example 7 Evaluation of Osteochondral Allograft Viability DuringPreservation at 25° C.

Osteochondral allografts (OCA) allow transplantation of viable,functional tissue for treatment of cartilage defects without the needfor immunosuppression. OCAs are reported to be successful in >75% ofcases when used for treatment of focal femoral condyle lesions. Thisstudy was designed to evaluate the ability of biomarkers and the mediaadditive to differentiate OCAs with low viability during culture usingvarious tissue preservation protocols at 25° C.

Methods Tissue Harvest and Culture:

During the course of two studies, medial and lateral femoral condyles(FC) from both knees of 14 adult canine cadavers were asepticallyharvested within 4 hours of euthanasia performed for reasons unrelatedto this study. The FCs were separated into one of 5 test groups based ondifferent media composition (M-1 (DMEM, 1×ITS (insulin, transferrin, andselenous acid), L-glutamine (20 mM), non-essential amino acids (1 mM),sodium pyruvate (10 mM), L-ascorbic acid (50 μg/ml), penicillin,streptomycin, and amphotericin B), M-2 (DMEM, 1×ITS (insulin,transferrin, and selenous acid), L-glutamine (20 mM), non-essentialamino acids (1 mM), sodium pyruvate (10 mM), L-ascorbic acid (50 μg/ml),penicillin, streptomycin, amphotericin B, and sodium borate (250μg/ml)), M-3 (DMEM, 1×ITS (insulin, transferrin, and selenous acid),L-glutamine (20 mM), non-essential amino acids (1 mM), sodium pyruvate(10 mM), L-ascorbic acid (50 μg/ml), penicillin, streptomycin,amphotericin B, and dexamethasone (0.01 μM))) and container condition(C-1, C-2, C-3) such that each FC from a single animal was placed in adistinct group. The following five media and container conditiongroupings were assessed for this study: M-1/C-1, M-1/C-2, M-1/C-3,M-2/C-1, and M-3/C-3. Tissues were stored at 25° C. without CO2supplementation in 60 mls of media for at least 63 days and up to 75days. The media were changed every 7 days and saved for biomarkeranalyses. On the next to last day of storage, 6 mls of the cellviability media additive as added to each sample and incubated for 24hours. After 24 hours, a media sample was analyzed for level offluorescence at a standard sensitivity. Increased fluorescence in themedia is indicative of cell metabolism and viability.

Media Analysis:

Media were analyzed for VEGF, matrix metalloproteinase (MMP)-2, -3, -9,and -13 (R&D System); and IL-6, IL-8, KC, and MCP-1 by Luminex multiplexassay (Millipore).

Tissue Viability Analysis:

Cartilage tissue was analyzed for cell viability using a fluorescentlive/dead assay (Invitrogen) and fluorescent microscopy. Images weretaken at 4× magnification using an Olympus F-View II camera andMicroSuite Basic Edition software. Greenstaining live cells weremanually counted, and the area of the tissue analyzed was determinedusing MicroSuite Basic Edition. The area of the tissue section analyzedwas measured, and the ratio of live cells (LC)/area (μm2) wasdetermined.

Data Analysis:

Data from the last day of storage were compared by Pearsonproduct-moment correlation using SigmaStat. Since the M-3 media wasdesigned to decrease tissue inflammation, the cytokines and chemokinesanalyzed in this study were significantly lower in this group comparedto all others during the course of storage. Therefore, the M-3 mediacytokine data could not be analyzed with the other media compositionsused in this study, but the MMP and media supplement data were used foranalysis.

Results Tissue Viability:

The mean tissue viability represented the viability of each group well,but each group had one outlier except the M-2/C-1 group and the M-3/C-3group. Further, the viabilities of M-1/C-3 and M-3/C-3 groups weresignificantly higher than all other groups.

Correlation Analysis (FIG. 13):

A significantly (p<0.001) moderate to strong positive correlation totissue viability was found for the media viability additive (r=0.724),IL-8 (r=0.598), VEGF (r=0.655), KC (r=0.738), MCP-1 (r=0.822), MMP-2(r=0.699), and MMP-3 (r=0.682). There was not a significant correlationto tissue viability for IL-6 (r=0.385, p=0.0694) and MMP-13 (r=0.203,p<0.319).

Discussion

These data indicate that similar to OCAs stored at 37 C, theconcentration of proteins in the preservation media at 25° C. have thepotential to act as biomarkers for identifying OCAs that have very lowcell viability and therefore are not considered suitable for clinicaluse.

1. A process for ligament tissue preservation comprising storing theligament tissue at room temperature in a container comprising culturemedia for from about 7 to about 70 days prior to implantation.
 2. Theprocess of claim 1, comprising testing the tissue for viability at leastonce prior to implantation in a patient.
 3. The process of claim 2,wherein testing for viability comprises assaying media withdrawn fromsaid container.
 4. The process of claim 2, wherein testing for viabilitycomprises adding a resazurin solution to the media and determining afluorescence level, wherein increased fluorescence indicates higher cellviability.
 5. The process of claim 1, comprising storing the tissue forfrom 29 to about 70 days.
 6. The process of claim 1 comprising changingsaid media at least once during the storing.
 7. The process of claim 6,comprising changing the media about once every two weeks during thestoring.
 8. The process of claim 1, wherein at least about 70% of cellsof said ligament tissue preserved by said process remain viable after 45days of storing.
 9. The process of claim 1, wherein the media is serumfree media.
 10. The process of claim 9, wherein the media comprisesDulbecco's Modified Eagle Medium (DMEM), high or low concentrations ofglucose, antibiotic compounds, antimycotic compounds, dexamethasone,ascorbate 2-phosphate, L-proline, sodium pyruvate, Transforming growthfactor-β3(TGF-β3), insulin, transferrin, and selenous acid.
 11. Theprocess of claim 1, wherein the ligament tissue comprises an allograft,the process comprising storing the ligament tissue in a tissuepreservation chamber comprising a base, lid, media inlet, and mediaoutlet; wherein the media inlet is coupled to at least a first filterfor maintaining a sterile environment inside the chamber; wherein thebase is configured to contain the ligament tissue and media; the outletextending into the chamber to permit removal of media; the media outletcomprising a one-way valve for exit of media from the chamber; whereinthe base is capable of receiving the lid to form a barrier tocontaminants.
 12. The process of claim 11, wherein the chamber comprisesa gas exchange port coupled to at least a first filter.
 13. The processof claim 12, wherein the media inlet, media outlet and gas exchange portare comprised within the lid.
 14. The process of claim 11, comprisingstoring the tissue in the chamber for from about 29 days to about 60days.
 15. The process of claim 1, wherein the tissue is maintained inserum-free media.
 16. The process of claim 15, wherein the mediacomprises an effective amount of dexamethasone.
 17. The process of claim1, wherein the ligament tissue comprises a section of spine, scapula,humerus, radius, ulna, pelvis, femur, tibia, patella, talus, phalangesor temporomandibular joint tissue.
 18. The process of claim 1, whereinthe ligament tissue comprises an allograft, the process comprisinglavaging of the ligament tissue in isotonic solution prior to storing.19. The process of claim 1, further comprising implanting the tissue ina subject in need thereof following said storing. 20-29. (canceled) 30.A method for preserving ligament tissue at room temperature in a chambercomprising culture media prior to implantation, the method comprising:placing the ligament tissue in a chamber base, the chamber baseconfigured to maintain the tissue and tissue preservation media; andforming a tissue preservation chamber by covering the chamber base witha lid to form a barrier to contaminants, the chamber comprising at leastone filter, a media inlet coupled to at least one filter for maintaininga sterile environment inside the chamber, and a media outlet, the mediaoutlet including a media outlet conduit that extends into the chamber topermit removal of media; wherein the media outlet comprises a one-wayvalve to prevent reentry of media exiting the chamber.
 31. The method ofclaim 30, wherein the chamber further comprises a gas exchange portcoupled to at least one filter.
 32. The method of claim 31, wherein thelid comprises the media inlet, media outlet, and gas exchange port. 33.The method of claim 30, comprising adding media to the chamber byforcing the through the media inlet and at least one filter.
 34. Themethod of claim 33, further comprising storing the tissue in the chamberfor from 29 days to about 70 days.
 35. The method of claim 34, furthercomprising removing media from the chamber through the media outlet. 36.The method of claim 34, further comprising simultaneously adding mediato the chamber by forcing through the media inlet and at least onefilter and removing media from the chamber through the media outlet. 37.The method of claim 34, further comprising applying tamper evident tapeto an interface between the chamber base and the lid.